Dysregulation of specific protein modifications in cells and/or tissues has been implicated in a wide range of pathophysiological conditions from neurodegeneration to heart failure. Progress in elucidating the role of these specific protein modifications in health and disease requires methods for identification and quantitation of the specific protein modifications, as well as the specific amino acids residues that are targets of protein modification.
For example, nitric oxide exerts a ubiquitous influence on cellular signaling, effected through the coordinated S-nitrosylation/denitrosylation of critical cysteine residues in multiple, functionally interrelated proteins. Accordingly, determining the role of S-nitrosylation in health and disease requires methods for identification and quantitation of S-nitrosothiols in protein, as well as the specific Cys residues that are the targets of S-nitrosylation.
The biotin switch technique (BST) has been widely adopted for assaying protein S-nitrosylation. However, the BST is labor intensive and is characterized by relatively low throughput, and thus is not well suited for proteomic analysis of S-nitrosothiols in protein. Furthermore, the BST is not easily adapted to modern proteomic techniques, such as isotopic labeling.
Accordingly, it is desirable to develop a means for the identification and quantitation of specific protein modifications that requires a minimum number of steps, is more economical than previously used methods, more efficiently detects higher molecular weight modified proteins, and is more easily combined with mass spectrometric methods.